The present invention relates generally to the field of medicine, and relates specifically to methods and compositions for modulating cell growth and death, including cell formation of tissues, using novel proteins, variants of these proteins and nucleic acids encoding them.
The integrity of the genome is of prime importance to a dividing cell. In response to DNA damage, eukaryotic cells rely upon a complex system of controls to delay cell-cycle progression. The normal eukaryotic cell-cycle is divided into 4 phases (sequentially G1, S, G2, M) which correlate with distinct cell morphology and biochemical activity. Cells withdrawn from the cell-cycle are said to be in G0, or non-cycling state. When cells within the cell-cycle are actively replicating, duplication of DNA occurs in the S phase, and active division of the cell occurs in M phase. See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 36, 1997). DNA is organized in the eukaryotic cell into successively higher levels of order that result in the formation of chromosomes. Non-sex chromosomes are normally present in pairs, and during cell division, the DNA of each chromosome replicates resulting in paired chromatids. (See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 5, 1997).
The eukaryotic cell cycle is tightly regulated by intrinsic mechanisms that ensure ordered progression through its various phases and surveillance mechanisms that prevent cycling in the presence of aberrant or incompletely assembled structures. These negative regulatory surveillance mechanisms have been termed checkpoints (Hartwell and Weinert, 1989, xe2x80x9cCheckpoints: controls that ensure the order of cell cycle eventsxe2x80x9d Science, 246: 629-634). The mitotic checkpoint prevents cells from undergoing mitosis until all chromosomes have been attached to the mitotic spindle whereas the DNA structure checkpoint, which can be subdivided into the replication and DNA damage checkpoint, result in arrests at various points in the cell cycle in the presence of DNA damage or incompletely replicated DNA (Elledge, 1996, xe2x80x9cCell cycle checkpoints: preventing an identity crisis.xe2x80x9d Science, 274: 1664-1672). These arrests are believed to allow time for replication to be completed or DNA repair to take place. Cell cycling in the presence of DNA damage, incompletely replicated DNA or improper mitotic spindle assembly can lead to genomic instability, an early step in tumorigenesis. Defective checkpoint mechanisms, resulting from inactivation of the p53, ATM, and Bub1 checkpoint gene products have been implicated in several human cancers.
Checkpoint delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase (Hartwell and Weinert, 1989, supra.). In many cases the DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases (Elledge, 1997, supra.). In human cells the DNA-damage induced G2 delay is largely dependent on inhibitory phosphorylation of Cdc2 (Blasina et al., 1997, xe2x80x9cThe role of inhibitory phosphorylation of cdc2 following DNA replication block and radiation induced damage in Human cells.xe2x80x9d Mol. Biol. Cell 8: 1013-1023; Jin et al., 1997, xe2x80x9cRole of inhibiting cdc2 phosphorylation in radiation-induced G2 arrest in human cells.xe2x80x9d J. Cell Biol. 134: 963-970), and is therefore likely to result from a change in the activity of the opposing kinases and phosphatases that act on Cdc2. However, evidence that the activity of these enzymes is substantially altered in response to DNA damage is lacking (Poon et al., 1997, xe2x80x9cThe role of cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells.xe2x80x9d Cancer Res., 57: 5168-5178).
Three distinct Cdc25 proteins are expressed in human cells. Cdc25A is specifically required for the G1-S transition (Hoffmann et al., 1994, xe2x80x9cActivation of the phosphatase activity of human CDC25A by a cdk2-cyclin E dependent phosphorylation at the G-1/S transition.xe2x80x9d EMBO J., 13: 4302-4310; Jinno et al., 1994, xe2x80x9cCdc25A is a novel phosphatase functioning early in the cell cyclexe2x80x9d EMBO J., 13: 1549-1556), whereas Cdc25B and Cdc25C are required for the G2-M transition (Gabrielli et al., 1996, xe2x80x9cCytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule mucleation in HeLa cellsxe2x80x9d J. Cell Sci., 109(5): 1081-1093; Galaktionov et al., 1991, xe2x80x9cSpecific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclinsxe2x80x9d Cell, 67: 1181-1194; Millar et al., 1991, xe2x80x9cp55CDC25 is a nuclear protein required for the initiation of mitosis in human cellsxe2x80x9d Proc. Natl. Acad. Sci. USA 88: 10500-10504; Nishijima et al., 1997, J. Cell Biol., 138: 1105-1116). The exact contribution of Cdc25B and Cdc25C to M-phase progression is not known.
Much of our current knowledge about checkpoint control has been obtained from studies using budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast. A number of reviews of our current understanding of cell cycle checkpoint in yeast and higher eukaryotes have recently been published (Hartwell and Kastan, 1994, xe2x80x9cCell cycle control and Cancerxe2x80x9d Science, 266: 1821-1828; Murray, 1994, xe2x80x9cCell cycle checkpointsxe2x80x9d Current Opinions in Cell Biology, 6: 872-876; Elledge, 1996, supra; Kaufmann and Paules, 1996, xe2x80x9cDNA damage and cell cycle checkpointsxe2x80x9d FASEB J., 10: 238-247). In the fission yeast six gene products, rad+, rad3+, rad9+, rad17+, rad26+, and hus1+ have been identified as components of both the DNA-damage dependent and DNA-replication dependent checkpoint pathways. In addition cds1+ has been identified as being required for the DNA-replication dependent checkpoint and rad27+/chk1+ has been identified as required for the DNA-damage dependent checkpoint in yeast.
Several of these genes have structural homologues in the budding yeast. Further conservation across eukaryotes has recently been suggested with the cloning of several human homologues of S. pombe checkpoint genes, including two related to S. pombe rad3+: ATM (ataxia telangiectasia mutated) (Savitsky et al., 1995, xe2x80x9cA single ataxia telangiectasia gene with a product similar to PI-3 kinasexe2x80x9d Science, 268: 1749-1753) and ATR (ataxia telangiectasia and rad3+ related)(Bentley et al, 1996, xe2x80x9cThe Schizosaccharomyces pombe rad3 checkpoint genesxe2x80x9d EMBO J., 15: 6641-6651; Cimprich et al., xe2x80x9ccDNA cloning and gene mapping of a candidate human cell cycle checkpoint proteinxe2x80x9d 1996, Proc. Natl. Acad. Sci. USA, 93: 2850-2855); and human homologues of S. pombe rad9+, Hrad9 (Lieberman et al., 1996, xe2x80x9cA human homolog of the Schizosaccharomyces pombe rad9+ checkpoint control genexe2x80x9d Proc. Natl. Acad. Sci. USA, 93: 13890-13895), Hrad1 (Parker et al., 1998, xe2x80x9cIdentification of a human homologue of the Schizosaccharomyces pombe rad17+ checkpoint genexe2x80x9d J. Biol. Chem. 273:18340-18346; Freire et al., 1998, xe2x80x9cHuman and mouse homologs of Schizosaccharomyces pombe rad1(+) and Saccharomyces cerevisia RAD17: linkage to checkpoint control and mammalian meiosisxe2x80x9d Genes Dev. 12:2560-2573; Udell et al., 1998, xe2x80x9cHrad1 and Mrad1 encode mammalian homologues of the fission yeast rad1(+) cell cycle checkpoint control genexe2x80x9d Nucleic Acids Res. 26:2971-3976), Hrad17 (Parker et al., 1998, supra), Hhus1 (Kostrub et al., 1998, xe2x80x9cHus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damagexe2x80x9d EMBO J. 17:2055-2066), Hchk1 (Sanchez et al., 1997, xe2x80x9cConservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25xe2x80x9d Science 277:1497-1501) and Hcds1 (Matusoka et al., 1998, xe2x80x9cLinkage of ATM to cell cycle regulation by the Chk2 protein kinasexe2x80x9d Science 282(5395): 1893-1897; Blasina et al., 1999, xe2x80x9cA human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatasexe2x80x9d Curr. Biology 9(1): 1-10).
Genetic and biochemical analysis of the checkpoint proteins in yeast and mammalian cells suggests that the checkpoint response is transmitted through a conventional signal transduction pathway. Hrad1, Hrad9, Hrad17, and Hhus1 transmit the signal emanating from damaged or incompletely replicated DNA to the central kinases ATM and ATR, which in turn activate the downstream kinases, Chk1 and Cds1. The DNA structure checkpoint responses ultimately lead to phosphorylation of the mitosis inducing phosphatase Cdc25 by Chk1 or Cds1. This phosphorylation event creates a binding site for 14-3-3 proteins that target Cdc25 for export from the nucleus to the cytoplasm, thus preventing it from removing an inhibitory phosphate from the cyclin dependent kinase, Cdc2. Removal of this inhibitory phosphate is required for passage from G2 to mitosis in every cell cycle. The DNA structure checkpoint responses prevent this from occurring and result in a G2/M arrest.
Whereas the Chk1 protein has been shown to be required for the G2/M DNA damage checkpoint in S. pombe, the replication checkpoint requires the activity of both Cds1 and Chk1. When replication is blocked by treatment with the ribonucleotide reductase inhibitor hydroxyurea (HU), wild type cells arrest prior to mitosis. A cds1chk1 double mutant fails to arrest in the presence of HU while both single mutants arrest normally (Russell, 1998, xe2x80x9cCheckpoints on the road to mitosisxe2x80x9d Trends in Biochemical Sciences 23(10):399-402). S. pombe Chk1 and Cds1 are both capable of phosphorylating Cdc25 and targeting it for binding by 14-3-3 proteins. Activation of the S. pombe Cds1 protein kinase by HU also results in enhanced binding to and phosphorylation of Weel, and accumulation of Mik1. These two protein kinases are required for the inhibitory phosphorylation of Cdc2 that prevents cells from entering mitosis suggesting an alternative to Cdc25C phosphorylation for checkpoint mediated cell cycle arrest. Recently, Cds1 has also been shown to be required for a DNA damage checkpoint in S-phase (Rhind and Russell, 1998, xe2x80x9cThe Schizosaccharomyces pombe S-phase checkpoint differentiates between different types of DNA damagexe2x80x9d Genetics 149(4): 1729-1737; Lindsay et al., 1998, xe2x80x9cS-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombexe2x80x9d Genes Dev. 12(3):382-395). A human homologue of S. pombe Cds1 that is activated by DNA damage and HU in an ATM-dependent manner and is capable of phosphorylating Cdc25C in vitro was recently identified (Matsuoka et al., 1998, supra; Blasina et al., 1999, supra). The human cDNA encodes a 543 amino acid protein which like its S. pombe homologue, contains a forkhead associated (FHA) domain N-terminal to the kinase domain. FHA domains are found in several other proteins including the S. cerevisiae Cds1 orthologue Rad53. Rad53 contains two FHA domains, one of which is required for interaction with the DNA damage checkpoint protein Rad9 in the presence of DNA damage (Sun et al., 1998, xe2x80x9cRad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpointxe2x80x9d Science 281(5374):272-274).
In order to develop new and more effective treatments and therapeutics for the amelioration of the effects of aging or disease such as cancer, it is important to identify and characterize mammalian, and in particular human, checkpoint proteins and to identify mediators of their activity. The present invention teaches the identification and characterization of human and murine nucleic acids encoding human Mus81 (Hmus81) and murine Mus81 (Mmus81) protein with significant homology to the S. pombe Mus81 protein that interacts with the S. pombe Cds1 FHA domain. The S. cerevisiae orthologue is reported to be involved in meiosis and DNA repair.
As described below, a Hmus81 gene acts as a checkpoint/repair gene and is involved with DNA repair. The checkpoint/repair delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase, and Hmus81 appears to act in both aspects, similarly to other known checkpoint/repair genes. In many cases, the DNA-damage response pathways will cause arrest, and the cell will fail to divide. However, a functional DNA repair mechanism will allow the damage to be corrected, and thus allow eventual cell division to occur.
In humans, excision repair is an important defense mechanism against two major carcinogens, sunlight and cigarette smoke. It has been found that individuals defective in excision repair exhibit a high incidence of cancer. (see Sancar, A, 1996, xe2x80x9cDNA Excision Repairxe2x80x9d Ann. Rev. Biochem. 65:43-81). Other mechanisms also act in a similar manner to repair DNA, such as mismatch repair which stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences. Inactivation of genes encoding these activities results in a large increase in spontaneous mutability and predisposition to tumor development. (see Modrich and Lahue, 1996, xe2x80x9cMismatch Repair in Replication Fidelity, Genetic Recombination and Cancer Biologyxe2x80x9d Ann. Rev. Biochem. 65: 101-33). The importance of maintaining fidelity in the DNA is amply illustrated by the many mechanisms for repair, and if unrepairable, arrest of cell division. (see Wood, R D, 1996, xe2x80x9cDNA Repair in Eukaryotesxe2x80x9d Ann. Rev. Biochem. 65:135-67).
Many chemotherapeutic agents are designed to disrupt or otherwise cause damage to the DNA of the targeted malignant cells. Antineoplastic agents such as alkylating agents, antimetabolites, and other chemical analogs and substances typically act by inhibiting nucleotide biosynthesis or protein synthesis, cross-linking DNA, or intercalating with DNA to inhibit replication or gene expression. Bleomycin and etoposide for example, specifically damage DNA and prevent repair.
The inhibition of Hmus81 gene or protein activity amplifies the potency of antineoplastic agents, and enhances the efficacy of their use as chemotherapeutic agents. This enhancement is beneficial in not only more thoroughly affecting the targeted cells, but by allowing for reduced dosages to be used in proportion to the increased efficacy, thus reducing unwanted side effects. Inhibition of Hmus81 or Mmus81 gene activity via anti-sense nucleic acid pharmaceuticals can be effected using the nucleic acids of the invention as the template for constructing the anti-sense nucleic acids. It is preferred to target the amino terminal end of the nucleic acid for anti-sense binding, and thus inhibition, as this reduces translation of the mRNA. Inhibition of Hmus81 protein activity can be effected by the use of altered or fragments of Hmus81 or Mmus81 protein to competitively inhibit the biochemical cascade that results in the repair of damaged DNA, or to cause cell arrest.
Disease can also result from defective DNA repair mechanisms, and include hereditary nonpolyposis colorectal cancer (defect in mismatch repair), Nijmegen breakage syndrome (defect in double strand break repair), Xeroderma pigmentosum, Cockayne syndrome, and Trocothiodystrophy (defect in nuclear excision repair). (see for example Lengauer et al., 1998, xe2x80x9cGenetic instabilities in human cancersxe2x80x9d Nature 396(6712):643-649; Kanaar et al., 1998, xe2x80x9cMolecular mechanisms of DNA double stranded repairxe2x80x9d Trends Cell Biol. 8(12):483-489).
It is further envisioned that the transient inhibition of Hmus81 gene or protein activity can be sufficient to effect improved treatment of cell behavior due to aging or disease. For example, the transient inhibition of DNA checkpoint/DNA damage arrest of cell division may allow the combined use of lower doses of chemotherapeutic agents to effect greater damage to targeted cells in the treatment of diseases such as cancer.
Novel genes and proteins encoded thereby are useful for modifying cell growth, division and death. One aspect of the invention is a novel mammalian, e.g., human or murine checkpoint/repair protein, the nucleic acids which encode for it and its protein variants, nucleic acid constructs, and methods for the production and use of mammalian Mus81 encoding gene and protein. As used herein, xe2x80x9ccheckpoint genexe2x80x9d means a gene which encodes for a protein which acts in the checkpoint/repair regulation of cell division. Such protein can effect both replication and DNA damage checkpoint activity, ie. having checkpoint/repair activity. Specific characterization of the mammalian Mus81 protein encoding nucleic acids and their role in cell cycle regulation provides for novel and useful compounds for modulating the mammalian cell cycle in a target cell.
As used herein, the terms xe2x80x9chuman Mus81 genexe2x80x9d, xe2x80x9cHmus81 encoding genexe2x80x9d and xe2x80x9cHmus81 genexe2x80x9d encompas human Mus81 encoding genes, including the allelic variants of the gene which will occur in a human population, but still encode for the same protein, splice variants of the gene, as well as the transcripts from such genomic genes, cDNA encoding for the transcript, and other nucleic acids which will encode a Hmus81 protein. As used herein, the terms xe2x80x9chuman Mus81 proteinxe2x80x9d, xe2x80x9cHmus81xe2x80x9d and xe2x80x9cHmus81 proteinxe2x80x9d refer generally to the protein expressed from a Hmus81 encoding nucleic acid, and includes splice variants and glycosylation variants of the protein which are generated by the translation and processing of the protein encoded by a Hmus81 encoding gene, and in particular to a human Mus81 protein having an amino acid sequence corresponding to that depicted as SEQ ID NO.: 2, 4, 8, and 10. In a preferred embodiment, the isolated nucleic acids of the invention correspond to a cDNA that encodes for a human Mus81 protein. Any particular isolated nucleic acid of the invention, preferably encodes for only one form of a human Mus81 protein.
As described in detail below, the human Mus81 encoding nucleic acids of the invention encompasses isolated nucleic acids comprising a nucleotide sequence corresponding to the nucleotide sequences disclosed herein and specifically identified as Human Mus811 (xe2x80x9cHmus81(1)xe2x80x9d; SEQ ID NO.: 1), Human Mus812 (xe2x80x9cHmus81(2)xe2x80x9d; SEQ ID NO.: 3), Human Mus813 (xe2x80x9cHmus81(3)xe2x80x9d; SEQ ID NO: 7), and Human Mus814 (xe2x80x9cHmus81(4)xe2x80x9d; SEQ ID NO: 9). All of the foregoing nucleic acids encode for a human Mus81 protein, and its equivalents. Thus, the present invention encompasses a nucleic acid having a nucleotide sequence which encodes for a Hmus81 protein and specifically encompasses a nucleotide sequence corresponding to the coding domain segment of the sequences that are depicted as SEQ ID NO.: 1, 3, 7, 9 and 25.
The present invention also encompasses a nucleic acid which encodes for two versions of Hmus81 protein having a nucleotide sequence corresponding to that depicted as SEQ ID NO.:25. This nucleic acid encodes for a Hmus81 protein having an amino acid residue sequence depicted as SEQ ID NO.: 4, wherein the 201 nucleotides from position 1274 to 1474 of the sequence of SEQ ID NO.: 25 containing a stop codon, have been deleted, thus allowing translation of the longer coding domain segment sequence of DNA. The nucleic acid having a corresponding nucleotide sequence as that depicted as SEQ ID NO.: 25 also encodes for the shorter Hmus81 protein having the amino acid sequence depicted as SEQ ID NO.: 2, from a shorter coding domain segment, leaving the intron in place.
Thus, in a preferred embodiment, the present invention encompasses nucleic acids which encode for human Mus81 proteins, and in particular, nucleic acids having a coding domain segment sequence corresponding to that represented by nucleotides 23-1675 of the nucleotide sequence depicted as SEQ ID NO.: 1; to that represented by nucleotides 185-1549 of the nucleotide sequence depicted as SEQ ID NO.:3; to that represented by nucleotides 26-1297 of the nucleotide sequence depicted as SEQ ID NO.:7; to that represented by nucleotides 26-1681 of the nucleotide sequence depicted as SEQ ID NO.:9; or as identified in SEQ ID NO.: 25.
The terms xe2x80x9cmurine Mus81 genexe2x80x9d and xe2x80x9cMmus81 genexe2x80x9d are used herein to refer to the novel murine Mus81 encoding genes. The terms xe2x80x9cmurine Mus81 proteinxe2x80x9d, xe2x80x9cMmus81xe2x80x9d and xe2x80x9cMmus81 proteinxe2x80x9d refer generally to the protein product of the Mmus81 genes and in particular, to murine Mus81 proteins having an amino acid residue sequence corresponding to that depicted as SEQ ID NO.: 12, 14, 16, and 18.
The terns xe2x80x9cmurine Mus81 genexe2x80x9d, xe2x80x9cMmus81 genexe2x80x9d and xe2x80x9cMmus81 encoding genexe2x80x9d encompass the Mmus81 genes, and in particular isolated nucleic acids comprising a nucleotide sequence corresponding to the nucleotide sequences disclosed herein and identified as Mouse (murine) Mus811 (xe2x80x9cMmus81(1)xe2x80x9d; SEQ ID NO.: 11), Mouse Mus812 (xe2x80x9cMmus81(2)xe2x80x9d; SEQ ID NO.: 13), Mouse Mus813 (xe2x80x9cMmus81(3)xe2x80x9d; SEQ ID NO: 15), and Mouse Mus814 (xe2x80x9cMmus81(4)xe2x80x9d; SEQ ID NO: 17), and the protein coding domain segments encoded for therein. In a preferred embodiment, the isolated nucleic acids of the invention correspond to a cDNA that encodes for a murine Mus81 protein. Any particular isolated nucleic acid of the invention, preferably encodes for only one form of a murine Mus81 protein.
In another preferred embodiment, the present invention encompasses nucleic acids which encode for murine Mus81 proteins, and in particular, nucleic acids which have a coding domain segment sequence corresponding to that represented by nucleotides 42-1694 of the nucleotide sequence depicted as SEQ ID NO.: 11; to that represented by nucleotides 15-1323 of the nucleotide sequence depicted as SEQ ID NO.: 13; to that represented by nucleotides 52-1644 of the nucleotide sequence depicted as SEQ ID NO.: 15; or to that represented by nucleotides 52-1614 of the nucleotide sequence depicted as SEQ ID NO.:17.
The present invention also encompasses nucleic acid constructs, vectors, plasmids, cosmids, retrovirus or viral constructs and the like which contain a nucleotide sequence encoding for a human Mus81 or murine Mus81 protein. In particular, the present invention provides for nucleic acid vector constructs which contain the nucleotide sequence of the Hmus81 coding domain segments of the nucleic acid depicted as SEQ ID NO.: 1, 3, 7, 9 or 25 and which are expressible as a protein. The present invention also provides for nucleic acid vector constructs which contain the Mmus81 coding domain segments of the nucleic acids depicted as SEQ ID NO.: 11, 13, 15, or 17.
The term xe2x80x9ctransgene capable of expressionxe2x80x9d as used herein means a suitable nucleotide sequence which leads to expression of Hmus81 or Mmus81 proteins, having the same function and/or the same or similar biological activity as such protein. The transgene can include, for example, genomic nucleic acid isolated from mammalian cells (e.g. human or mouse) or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state. Preferably, the transgene comprises the nucleotide sequence encoding the proteins according to the invention as described herein, or a biologically active portion of said protein. A biologically active protein should be taken to mean, and not limited to, a fusion product, fragment, digestion fragment, segment, domain or the like of a Mus81 protein having some if not all of the protein activity as a whole Mus81 protein. A biologically active protein thus contains a biologically functional portion of a mammalian Mus81 protein conveying a biochemical function thereof.
The present invention encompasses nucleic acid vectors that are suitable for the transformation of host cells, whether eukaryotic or prokaryotic, suitable for incorporation into viral vectors, or suitable for in vivo or in vitro protein expression. Particularly preferred host cells for prokaryotic expression of protein include, and are not limited to bacterial cells such as E. coli. Suitable host cells for eukaryotic expression of protein include, and are not limited to mammalian cells of human or murine origin and the like, or yeast cells. In a preferred embodiment, expression of protein, as described below, is accomplished by viral vector transformation of immortalized human cells.
The present invention further embodies a nucleotide sequence which encodes for a human Mus81 or murine Mus81 protein, in tandem with, or otherwise in conjunction with additional nucleic acids for the generation of fusion protein products. Human Mus81 fusion proteins will contain at least one segment of the protein encoded for by the nucleic acid depicted as the coding domain segment depicted in the nucleotide sequence described as SEQ ID NO.: 1, 3, 7, and 9. Similarly, murine Mus81 fusion protein will contain at least one segment of protein encoded for by the coding domain segments of the nucleic acid depicted as SEQ ID NO.: 11, 13, 15, and 17.
The present invention also encompasses isolated nucleic acids or nucleic acid vector constructs containing nucleic acid segments, adapted for use as naked DNA transformant vectors for incorporation and expression in target cells. Also provided are inhibitors of human Mus81 or murine Mus81 encoding nucleic acid transcripts, such as anti-sense DNA, triple-helix nucleic acid, double-helix RNA or the like. Biologically active anti-sense DNA molecule formulations are those which are the complement to the nucleotide sequence of the human Mus81 or murine Mus81 encoding genes or fragments thereof, whether complementary to contiguous or discontinuous portions of the targeted nucleotide sequence, and are inhibitors of the human Mus81 or murine Mus81 protein expression in cells. Such inhibitors and inhibition are useful for many purposes including and not limited to, in vitro analysis of the cell-cycle checkpoint pathway, detection and/or evaluation of inhibiting or potentiating compounds, and for in vivo therapy.
The present invention also provides for compositions incorporating modified nucleotides or substitute backbone components which encode for the nucleotide sequence of a human Mus81 or murine Mus81 encoding gene, or fragments thereof.
The present invention encompasses the use of anti-sense nucleic acids which comprise a nucleic acid that is the complement of at least a portion of a nucleic acid encoding for a human Mus81 or murine Mus81 protein. Also envisioned are biologically active analogs of this antisense molecule selected from the group consisting of peptide nucleic acids, methylphosphonates and 2-O-methyl ribonucleic acids. An antisense molecule of the invention can also be a phosphorothioate analog.
Also encompassed are pharmaceutical preparations for inhibiting Hmus81 protein expression or function in a cell which comprises an antisense nucleic acid analog which is capable of entering said cell and binding specifically to a nucleic acid molecule encoding for Hmus81 protein. The antisense nucleic acid is present in a pharmaceutically acceptable carrier and has a nucleotide sequence complementary to at least a portion of the nucleic acid of SEQ ID NO.: 1, 3, 7, 9 or 25. It is also envisioned that this pharmaceutical preparation can comprise a nucleic acid having a sequence complementary to at least the nucleotides encoding for amino acid residues 1-50 of the amino acid residue sequence of SEQ ID NO.: 2, 4, 8, or 10. In a preferred embodiment, the pharmaceutical preparation comprises a nucleic acid having a nucleotide sequence complementary to at least nucleotides 1-20 of a coding domain segment in the nucleotide sequence depicted as SEQ ID NO.: 1, 3, 7, 9 or 25. In a most preferred embodiment, the antisense nucleic acid comprises a nucleic acid having a sequence complementary to at least nucleotides 1-10 of a coding domain segment in the nucleotide sequence depicted as SEQ ID NO.: 1, 3, 7, 9 or 25.
The present invention also encompasses nucleotide sequences which would encode for the Hmus81 protein having an amino acid sequence as that depicted by that of SEQ ID NO.: 2, 4, 8 or 10 based upon synonymous codon substitution given the knowledge of the triplet codons and which amino acids they encode, based upon the coding domain segment of the nucleotide sequence depicted in SEQ ID NO. 1, 3, 7, 9 or 25. The equivalent synonymous nucleic acid code for generating any nucleotide sequence which will encode for a protein having a particular amino acid sequence is known and predictable to one of skill in the art.
In a preferred embodiment codon usage is optimized to increase protein expression as desired for the target host cell, such as where a nucleic acid is modified so that it comprises a protein coding domain segment of the nucleotide sequence depicted in SEQ ID NO.: 1, 3, 7, 9, 11, 13, 15, 17 or 25, wherein the least preferred codons are substituted with those that are most preferred in the target host cell. In the case of human target host cells, the least preferred codons are ggg, att, ctc, tcc, and gtc.
The invention also provides for methods of generating human Mus81 or murine Mus81 protein, fusion proteins, or fragments thereof by using recombinant DNA technology and the appropriate nucleic acid encoding for human Mus81 or murine Mus81 protein. The invention provides for incorporating an appropriate nucleotide sequence into a suitable expression vector, the incorporation of suitable control elements such as a ribosome binding site, promoter, and/or enhancer element, either inducible or constitutively expressed. The invention provides for the use of expression vectors with or without at least one additional selectable marker or expressible protein. The invention provides for methods wherein a suitably constructed expression vector is transformed or otherwise introduced into a suitable host cell, and protein is expressed by such a host cell. The present invention also provides transformed host cells, which are capable of producing human Mus81 or murine Mus81 protein, fusion protein, or fragments thereof The expression vector including said nucleic acid according to the invention may advantageously be used in vivo, such as in, for example, gene therapy.
The invention encompasses mammalian, e.g. human or murine Mus81 protein, fusion products, and biologically active portions thereof produced by recombinant DNA technology and expressed in vivo or in vitro. A biologically active portion of a protein is protein segment or fragment having the enzymatic activity of, or at least a some enzymatic activity of the whole mammalian Mus81 protein, when compared under similar conditions. For example, it will be readily apparent to persons skilled in the art that nucleotide substitutions or deletions may be introduced using routine techniques, which do not affect the protein sequence encoded by said nucleic acid, or which encode a biologically active, functional protein according to the invention. Manipulation of the protein to generate fragments as a result of enzyme digestion, or the modification of nucleic acids encoding for the protein can similarly result in biologically active portions of the mammalian Mus81 protein.
Complete protein, fusion products and biologically active portions thereof of the mammalian Mus81 protein are useful for therapeutic formulations, diagnostic testing, and as immunogens, as for example to generate antibodies thereto. The invention thus encompasses Hmus81 and Mmus81 protein produced by transformed host cells in small-scale or large-scale production. The invention encompasses complete Hmus81 and Mmus81 protein, in either glycosylated or unglycosylated forms, produced by either eukaryotic or prokaryotic cells. The present invention provides for Hmus81 and Mmus81 protein expressed from mammalian, insect, plant, bacterial, fungal, or any other suitable host cell using the appropriate transformation vector as known in the art. The present invention encompasses Hmus81 and Mmus81 protein that is produced as a fusion protein product, conjugated to a solid support, or Hmus81 and Mmus81 protein which is labeled with any chemical, radioactive, fluorescent, chemiluminescent or otherwise detectable marker.
The present invention also provides Hmus81 and Mmus81 proteins isolated from natural sources and enriched in purity over that found in nature. Also provided are pharmaceutical formulations of Hmus81 and Mmus81 protein as well as formulations of the Hmus81 and Mmus81 protein in pharmaceutically acceptable carriers or excipients.
The present invention also encompasses the use of human Mus81 or murine Mus81 protein, fusion protein, or biololgically active fragments thereof to generate specific antibodies which bind specifically to the human Mus81 or murine Mus81 protein, or both, as either polyclonal or monoclonal antibodies generated by the immunization of a mammal with human Mus81 protein having the amino acid residue sequence, or an immunogenic fragment of the amino acid residue sequence shown as SEQ ID NO.: 2, 4, 8, or 10, or the murine Mus81 protein having the amino acid residue sequence shown as SEQ ID NO.: 12, 14, 16 or 18. An immunogenic fragment is one which will elicit an immune response, when injected into a immunologically competent host under immunogenic conditions, and generate antibodies specific for the immunogenic fragment.
The present invention also encompasses equivalent proteins where substitutions of amino acids for amino acid residues as shown in the amino acid sequence encoding for human Mus81 protein (SEQ ID NO.: 2, 4, 8, 10) or murine Mus81 protein (SEQ ID NO.: 12, 14, 16, 18) are made. Such amino acid substitutions include conservative substitutions of similar amino acid residues that are reasonably predictable as being equivalent, or semi-conservative substitutions which have a reasonably predictable effect on solubility, glycosylation, or protein expression. For example, non-polar (hydrophobic side-chain) amino acids alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; uncharged polar amino acids glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; charged polar amino acids aspartic acid, glutamic acid; basic amino acids lysine, arginine, and histidine are understood by those in the art to have functionally predictable effects when substituted. Amino acid substitutions also include replacement of amino acid residues with modified amino acid residues or chemically altered substitutes.
The present invention also encompasses nucleic acids which encode for such equivalent proteins and the embodiments thereof which encode for the human Mus81 proteins or murine Mus81 proteins. Specific modification can be made of codons used in the nucleic acids corresponding to the human Mus81 or murine Mus81 encoding genes of the invention such that the modified nucleic acids utilize codons preferred by the target host cell, while still encoding for the human Mus81 or murine Mus81 protein. This can be accomplished by conservative synonymous codon substitutions that reduce the number of less preferred codons and/or an increase in the number of preferred codons used by the target host cell The present invention also encompasses modified nucleic acids which incorporate, for example, intemucleotide linkage modification, base modifications, sugar modification, nonradioactive labels, nucleic acid cross-linking, and altered backbones including PNAs (polypeptide nucleic acids).
The knowledge that Hmus81 acts as a checkpoint/repair protein and is most likely involved in DNA repair, allows for the use of the compounds of the invention in therapeutic treatment of diseases which involve abnormal DNA damage checkpoint/repair function, or that would advantageously inhibit DNA repair in a targeted cell. The present invention further provides for the use of the compounds of the present invention as therapeutics for the treatment of cancer. In one embodiment, inhibitors or agents which inhibit the function of the normal proteins and/or genes of the invention would be useful to sensitize cells for treatment with chemotherapeutics, radiation, DNA damaging agents, or replication inhibitors.
The present invention also encompasses methods for screening test compounds for efficacy in effecting the Mus81 mediated checkpoint/repair function of eukaryotic cells. These methods comprise contacting a test compound to eukaryotic cells, and detecting any change in mammalian Mus81 expression or function. Also encompassed are methods of screening wherein a compound is administered, and detection of change in Hmus81 or Mmus81 gene expression or function is accomplished by assaying for Hmus81 or Mmus81 mRNA production or by assaying for Hmus81 or Mmus81 protein expression. Methods for detection of changes in expression level of a particular gene are known in the art. In particular, the present invention allows for the screening of candidate substances for efficacy in modifying the mammalian Mus81 mediated DNA damage checkpoint/repair or DNA repair function by screening for any change in nuclease, phosphorylation or kinase activity of mammalian Mus81 protein. The compounds or substances identified by the assays of the invention, or compounds corresponding to such compounds or substances, can be used for the manufacture of pharmaceutical therapeutics.
Methods of identifying a chemical compound that modulates the Mus81 dependent cell cycle pathway are provided for as well. Such methods comprise administering the chemical compound to be tested to a host cell, and detecting the amount of mammalian Mus81 protein in said cell, and comparing the amount detected with that of a normal untreated cell. Further provided for is a method of identifying a chemical compound that modulates the Mus81 dependent cell cycle pathway, which method comprises administering the chemical compound to be tested to a biochemical mixture of Hmus81 protein and a suitable substrate, and detecting the level of Hmus81 protein activity in said mixture, and comparing the detected activity with that of a normal untreated biochemical mixture of Hmus81 protein. As shown in the examples below, isolated Hmus81 protein and suitable substrates can be measured in isolated chemical reactions.
In one embodiment, the present invention also provides for pharmaceutical compositions which comprise the Hmus81 protein, Hmus81 nucleic acid, or Hmus81 anti-sense nucleic acids. The therapeutic Hmus81 protein can be normally glycosylated, modified, or unglycosylated depending upon the desired characteristics for the protein. Similarly, Hmus81 protein includes the complete long or short protein, fusion product, or functional or immunogenic fragment thereof. Hmus81 nucleic acids include those encoding for the entire long or short protein, portions of the protein, fusion protein products, and fragments thereof. Also included are modified forms of nucleic acids including those incorporating substitute base analogs, modified bases, PNAs and those incorporating preferred codon usage. Anti-sense nucleic acids include complementary nucleic acids which can bind specifically to the targeted nucleic acids, having full, part or discontinuous segments of complementary nucleic acid which can be DNA, RNA or analog compounds thereof In another embodiment, the present invention provides for compounds or substances identified as suitable for use as a therapeutic in pharmaceutical formulations by the assays of the invention. These pharmaceutical compositions can further include chemotherapeutic agents for the use in treating cancer, or be administered in a regimen coordinated with the administration of other anti-cancer therapies. The present invention, in one embodiment, encompasses methods for combined chemotherapy using the Hmus81 derived pharmaceuticals independently, and in combination with other chemotherapeutic agents, and in a second embodiment as admixtures with other anti-cancer therapeutics for single dose administration.
Similarly, murine Mus81 protein, or nucleic acids encoding for the protein can be used to modulate the cell cycle of murine or non-murine mammalian cells. Nucleic acids encoding for the murine Mus81 protein, can be used to produce murine Mus81 protein by recombinant means for use in pharmaceuticals, detection methods and kits, and assay systems in the same manner as human Mus81 protein.
The invention provides for a transgenic cell, transformed cell, tissue or organism comprising a transgene capable of expressing human Mus81 protein, which protein comprises the amino acid sequence illustrated in FIG. 1A (SEQ ID NO.:2), FIG. 1B (SEQ ID NO.:4), FIG. 1C (SEQ ID NO.:8), FIG. 1D (SEQ ID NO.: 10), or a murine Mus81 protein, which protein comprises the amino acid sequence illustrated in FIG. 2A (SEQ ID NO.: 12), FIG. 2B (SEQ ID NO.: 14), FIG. 2C (SEQ ID NO.: 16), FIG. 2D (SEQ ID NO.:18), or the amino acid sequence of a biologically active functional equivalent or bioprecursor or biologically active fragment therefor. And for the isolated protein produced by such transformed host cells.